Systems and methods for real-time PCR

ABSTRACT

In one aspect, the present invention provides a systems and methods for the real-time amplification and analysis of a sample of DNA.

This application claims the benefit of Provisional Patent ApplicationNo. 60/806,440, filed on Jun. 30, 2006, which is incorporated herein bythis reference.

This application is related to patent application Ser. No. 11/505,358,filed on Aug. 17, 2006, which is incorporated herein by this reference.

BACKGROUND

1. Field of the Invention

The present invention relates to methods for amplifying nucleic acids inmicro-channels. In some embodiments, the present invention relates tomethods for performing a real-time polymerase chain reaction (PCR) in acontinuous-flow microfluidic system and to methods for monitoringreal-time PCR in such systems.

2. Discussion of the Background

The detection of nucleic acids is central to medicine, forensic science,industrial processing, crop and animal breeding, and many other fields.The ability to detect disease conditions (e.g., cancer), infectiousorganisms (e.g., HIV), genetic lineage, genetic markers, and the like,may facilitate disease diagnosis and prognosis, marker assistedselection, correct identification of crime scene features, the abilityto propagate industrial organisms and many other techniques.Determination of the integrity of a nucleic acid of interest can berelevant to the pathology of an infection or cancer.

One of the most powerful and basic technologies to detect smallquantities of nucleic acids is to replicate some or all of a nucleicacid sequence many times, and then analyze the amplification products.PCR is perhaps the most well-known of a number of differentamplification techniques.

PCR is a powerful technique for amplifying short sections of DNA. WithPCR, one can quickly produce millions of copies of DNA starting from asingle template DNA molecule. PCR includes a three phase temperaturecycle of denaturation of DNA into single strands, annealing of primersto the denatured strands, and extension of the primers by a thermostableDNA polymerase enzyme. This cycle is repeated so that there are enoughcopies to be detected and analyzed.

In principle, each cycle of PCR could double the number of copies. Inpractice, the multiplication achieved after each cycle is always lessthan 2. Furthermore, as PCR cycling continues, the buildup of amplifiedDNA products eventually ceases as the concentrations of requiredreactants diminish.

For general details concerning PCR, see Sambrook and Russell, MolecularCloning—A Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (2000); Current Protocols inMolecular Biology, F. M. Ausubel et al., eds., Current Protocols, ajoint venture between Greene Publishing Associates, Inc. and John Wiley& Sons, Inc., (supplemented through 2005) and PCR Protocols A Guide toMethods and Applications, M. A. Innis et al., eds., Academic Press Inc.San Diego, Calif. (1990).

Real-time PCR refers to a growing set of techniques in which onemeasures the buildup of amplified DNA products as the reactionprogresses, typically once per PCR cycle. Monitoring the accumulation ofproducts over time allows one to determine the efficiency of thereaction, as well as to estimate the initial concentration of DNAtemplate molecules. For general details concerning real-time PCR seeReal-Time PCR: An Essential Guide, K. Edwards et al., eds., HorizonBioscience, Norwich, U.K. (2004).

Several different real-time detection chemistries now exist to indicatethe presence of amplified DNA. Most of these depend upon fluorescenceindicators that change properties as a result of the PCR process. Amongthese detection chemistries are DNA binding dyes (such as SYBR® Green)that increase fluorescence efficiency upon binding to double strandedDNA. Other real-time detection chemistries utilize Foerster resonanceenergy transfer (FRET), a phenomenon by which the fluorescenceefficiency of a dye is strongly dependent on its proximity to anotherlight absorbing moiety or quencher. These dyes and quenchers aretypically attached to a DNA sequence-specific probe or primer. Among theFRET-based detection chemistries are hydrolysis probes and conformationprobes. Hydrolysis probes (such as the TaqMan® probe) use the polymeraseenzyme to cleave a reporter dye molecule from a quencher dye moleculeattached to an oligonucleotide probe. Conformation probes (such asmolecular beacons) utilize a dye attached to an oligonucleotide, whosefluorescence emission changes upon the conformational change of theoligonucleotide hybridizing to the target DNA.

A number of commercial instruments exist that perform real-time PCR.Examples of available instruments include the Applied Biosystems PRISM7500, the Bio-Rad iCylcer, and the Roche Diagnostics LightCycler 2.0.The sample containers for these instruments are closed tubes whichtypically require at least a 10 μl volume of sample solution. If thelowest concentrations of template DNA detectable by a particular assaywere on the order of one molecule per microliter, the detection limitfor available instruments would be on the order of tens of targets persample tube. Therefore, in order to achieve single molecule sensitivity,it is desirable to test smaller sample volumes, in the range of 1-1000nl.

More recently, a number of high throughput approaches to performing PCRand other amplification reactions have been developed, e.g., involvingamplification reactions in microfluidic devices, as well as methods fordetecting and analyzing amplified nucleic acids in or on the devices.Thermal cycling of the sample for amplification is usually accomplishedin one of two methods. In the first method, the sample solution isloaded into the device and the temperature is cycled in time, much likea conventional PCR instrument. In the second method, the sample solutionis pumped continuously through spatially varying temperature zones.

For example, Lagally et al. (Anal Chem 73:565-570 (2001)) demonstratedamplification and detection of single template DNA in a 280 nl PCRchamber. Detection of products was made post-PCR using capillaryelectrophoresis. On the other hand, Kopp et al. (Science 280:1046-1048(1998)) demonstrated continuous-flow PCR using a glass substrate with aserpentine channel passing over three constant temperature zones at 95°C. (denature), 72° C. (extension), and 60° C. (annealing). The 72° C.zone was located in the central region and had to be passed throughbriefly in going from 95° C. to 60° C.

Detection was made post-PCR using gel electrophoresis. Since this PCRtechnique is not based on heating the entire surfaces of the reactionvessel, the reaction rate is determined by a flow rate, not aheating/cooling rate. Neither of these references described real-timemonitoring of the PCR reaction.

Park et al. (Anal Chem 75:6029-6033 (2003)) describe a continuous-flowPCR device that uses a polyimide coated fused silica capillary wrappedinto a helix around three temperature-controlled blocks. Sample volumeswere 2 μl. Detection was made post PCR using gel electrophoresis.Reference was made to the possibility of adapting their instrument forreal-time PCR by using a capillary coated with PTFE instead ofnon-transparent polyimide. See also, Hahn et al. (WO 2005/075683).

Enzelberger et al. (U.S. Pat. No. 6,960,437) describe a microfluidicdevice that includes a rotary channel having three temperature zones. Anumber of integrated valves and pumps are used to introduce the sampleand to pump it through the zones in a rotary fashion.

Knapp et al. (U.S. Patent Application Publication No. 2005/0042639)describe a microfluidic device capable of single molecule amplification.A planar glass device with several straight parallel channels isdisclosed. A mixture of target DNA and PCR reagents is injected intothese channels. In a first embodiment, the channels are filled with thismixture and flow is stopped. Then the entire length of the channels isthermally cycled. After thermal cycling is completed, the channels areimaged in order to detect regions of fluorescence where DNA has beenamplified. In a second embodiment, the PCR mixture flows continuouslythrough the amplification zone as the temperature is cycled, andfluorescence is detected downstream of the amplification zone. Differentdegrees of amplification are achieved by altering the time spent incycling, through changing distance traveled under cycling, and the like.It is worth noting that this method varies conditions (such as cyclesexperienced) for separate consecutive sample elements, rather thanmonitoring the progress of individual sample elements over time.

A need exists for robust, high throughput methods of real-time PCR thatcan be performed efficiently and accurately.

SUMMARY

The present invention relates to systems and methods for amplifyingnucleic acids in micro-channels. In some embodiments, the presentinvention relates to systems and methods for performing a real-timepolymerase chain reaction (PCR) in a continuous-flow microfluidic systemand to systems and methods for monitoring real-time PCR in such systems.

Thus, in a first aspect, the present invention provides a system forperforming real-time PCR and melting of DNA. In some embodiments, thesystem includes: a microfluidic device comprising: a microfluidicchannel having a PCR zone and a DNA melting zone, a first reagent wellin fluid communication with the channel and a second reagent well influid communication with the channel; a sipper connected to themicrofluidic device, the sipper being in fluid communication with thechannel; a pump for forcing a sample to flow through the channel; a wellplate comprising a buffer well for storing a buffer solution and asample well for storing a sample solution containing a DNA sample; abuffer solution storage container for storing a buffer solution, thebuffer solution storage container being in fluid communication with thebuffer well of the well plate; a positioning system operable to positionthe well plate; a temperature control system for cycling the temperatureof a sample while the sample flows through the PCR zone of the channeland for providing heat for melting the DNA contained in the sample whilethe sample flows through the DNA melting zone; an imaging system fordetecting emissions from the PCR zone and from the DNA melting zone, theimaging system comprising: an excitation source and a detectorconfigured and arranged to detect emissions from the PCR zone and/or theDNA melting zone; and a main controller in communication with (a) thetemperature control system, (b) the positioner, (c) the imaging system,and (d) a display device.

In a second aspect, the present invention provides a method forperforming real-time PCR and melting of DNA. In some embodiments, themethod includes: creating a script, wherein the script containsconfiguration information; preparing a microfluidic device having amicrofluidic channel having a PCR zone and a DNA melting zone; readingthe script to obtain the configuration information; positioning a wellplate relative to the device, the well plate having a buffer wellcontaining a buffer solution and sample well containing a samplesolution containing a DNA sample; activating a pump, wherein the pump isconfigured to create a pressure differential that causes the samplesolution to flow through the channel, wherein the sample flows throughthe PCR zone prior to flowing through the DNA melting zone; while thesample is flowing through the channel: cycling the temperature of thesample according to configuration information included in the script asthe sample flows through the PCR zone to amplify the DNA sample;obtaining images of the sample as the sample flows through the PCR zone;processing the images; and melting the amplified DNA.

The above and other embodiments of the present invention are describedbelow with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and form partof the specification, illustrate various embodiments of the presentinvention. In the drawings, like reference numbers indicate identical orfunctionally similar elements.

FIG. 1 is a block diagram illustrating a system according to someembodiments of the invention.

FIG. 2 is a block diagram illustrating a temperature control systemaccording to some embodiments of the invention.

FIG. 3 is a block diagram illustrating an imaging system according tosome embodiments of the invention.

FIG. 4 is a flow chart illustrating a process according to someembodiments of the invention.

FIG. 5 is a flow chart illustrating a process according to someembodiments of the invention.

FIG. 6 is a flow chart illustrating a process according to someembodiments of the invention.

FIG. 7 is a flow chart illustrating a process according to someembodiments of the invention.

FIG. 8 is a flow chart illustrating a process according to someembodiments of the invention.

FIG. 9 is a flow chart illustrating a process according to someembodiments of the invention.

FIG. 10 is a flow chart illustrating a process according to someembodiments of the invention.

FIG. 11 is a flow chart illustrating a process according to someembodiments of the invention.

FIG. 12 illustrates an image of a DNA melting zone.

FIG. 13 illustrates a user defining regions of interest.

FIG. 14 illustrates a pixel window.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As used herein, the words “a” and “an” mean “one or more.”

The present invention provides systems and methods for real time PCR andhigh resolution thermal melt. Referring to FIG. 1, FIG. 1 illustrates afunctional block diagram of a system 100 according to some embodimentsof the invention. As illustrated in FIG. 1, system 100 may include amicrofluidic device 102. Microfluidic device 102 may include one or moremicrofluidic channels 104. In the examples shown, device 102 includestwo microfluidic channels, channel 104 a and channel 104 b. Althoughonly two channels are shown in the exemplary embodiment, it iscontemplated that device 102 may have fewer than two or more than twochannels. For example, in some embodiments, device 102 includes eightchannels 104.

Device 102 may include two DNA processing zones, a DNA amplificationzone 131 (a.k.a., PCR zone 131) and a DNA melting zone 132. A DNA sampletraveling through the PCR zone 131 may undergo PCR, and a DNA samplepassing through melt zone 132 may undergo high resolution thermalmelting. As illustrated in FIG. 1, PCR zone 131 includes a first portionof channels 104 and melt zone 132 includes a second portion of channels104, which is down stream from the first portion.

Device 102 may also include a sipper 108. Sipper 108 may be in the formof a hollow tube. Sipper 108 has a proximal end that is connected to aninlet 109 which inlet couples the proximal end of sipper 108 to channels104.

Device 102 may also include a common reagent well 106 which is connectedto inlet 109. Device 102 may also include a locus specific reagent well105 for each channel 104. For example, in the embodiment shown, device102 includes a locus specific reagent well 105 a, which is connected tochannel 104 a, and may include a locus specific reagent well 105 b whichis connected to channel 104 b. Device 102 may also include a waste well110 for each channel 104.

The solution that is stored in the common reagent well 106 may containdNTPs, polymerase enzymes, salts, buffers, surface-passivating reagents,one or more non-specific fluorescent DNA detecting molecules, a fluidmarker and the like. The solution that is stored in a locus specificreagent well 105 may contain PCR primers, a sequence-specificfluorescent DNA probe or marker, salts, buffers, surface-passivatingreagents and the like.

In order to introduce a sample solution into the channels 104, system100 may include a well plate 196 that includes a plurality of wells 198,at least some of which contain a sample solution (e.g., a solutioncontaining a nucleic acid sample). In the embodiment shown, well plate196 is connected to a positioning system 194 which is connected to amain controller 130.

Main controller 130 may be implemented using a PXI-8105 controller whichis available from National Instruments Corporation of Austin, Tex.Positioning system 194 may include a positioner (e.g., the MX80positioner available from Parker Hannifin Corporation of PA (“Parker”))for positioning well plate 196, a stepping drive (e.g., the E-ACMicrostepping Drive available from Parker) for driving the positioner,and a controller (e.g., the 6K4 controller available from Parker) forcontrolling the stepping drive.

To introduce a sample solution into the channels 104, the positioningsystem 194 is controlled to move well plate 196 such that the distal endof sipper 108 is submerged in the sample solution stored in one of thewells 198. FIG. 1 shows the distal end of 108 being submerged within thesample solution stored in well 198 n.

In order to force the sample solution to move up the sipper and into thechannels 104, a vacuum manifold 112 and pump 114 may be employed. Thevacuum manifold 112 may be operably connected to a portion of device 102and pump 114 may be operably connected to manifold 112. When pump 114 isactivated, pump 114 creates a pressure differential (e.g., pump 114 maydraw air out of a waste well 110), and this pressure differential causesthe sample solution stored in well 198 n to flow up sipper 108 andthrough inlet channel 109 into channels 104. Additionally, this causesthe reagents in wells 106 and 105 to flow into a channel. Accordingly,pump 114 functions to force a sample solution and real-time PCR reagentsto flow through channels 104. As illustrated in FIG. 1, melt zone 132 islocated downstream from PCR zone 131. Thus, a sample solution will flowfirst through the PCR zone and then through the melting zone.

Referring back to well plate 196, well plate 196 may include a buffersolution well 198 a. In one embodiment, buffer solution well 198 a holdsa buffer solution 197. Buffer solution 197 may comprise a conventionalPCR buffer, such as a conventional real-time (RT) PCR buffer.Conventional PCR buffers are available from a number of suppliers,including: Bio-Rad Laboratories, Inc., Applied Biosystems, RocheDiagnostics, and others.

In order to replenish buffer solution well 198 a with the buffersolution 197, system 100 may include a buffer solution storage container190 and a pump 192 for pumping the buffer solution 197 from container190 into well 198 a. Additionally, pump 192 may be configured to notonly add solution 197 to well 198 a, but also remove solution 197 fromwell 198 a, thereby re-circulating the solution 197.

In order to achieve PCR for a DNA sample flowing through the PCR zone131, the temperature of the sample must be cycled, as is well known inthe art. Accordingly, in some embodiments, system 100 includes atemperature control system 120. The temperature control system 120 mayinclude a temperature sensor, a heater/cooler, and a temperaturecontroller. In some embodiments, a temperature control system 120 isinterfaced with main controller 130 so that main controller 130 cancontrol the temperature of the samples flowing through the PCR zone andthe melting zone.

Main controller 130 may be connected to a display device for displayinga graphical user interface. Main controller 130 may also be connected touser input devices 134, which allow a user to input data and commandsinto main controller 130.

To monitor the PCR process and the melting process that occur in PCRzone 131 and melt zone 132, respectively, system 100 may include animaging system 118. Imaging system 118 may include an excitation source,an image capturing device, a controller, and an image storage unit.

Referring now to FIG. 2, FIG. 2 illustrates the temperature controlsystem 120 according to some embodiments of the invention. Asillustrated in FIG. 2, temperature control system 120 may include anumber of heating and/or cooling devices (e.g., a thermoelectric cooler(TEC), which is also known as a Peltier device, or other heating/coolingdevice), a number of temperature controllers, and a number oftemperature sensors.

In the embodiment shown, temperature control system 120 includes a TEC201 for heating and cooling inlet 109, a TEC 202 for heating and coolingthe PCR zone, a TEC 203 for heating and cooling the melting zone, and aTEC 204 for heating and cooling the waste well 110. Each TEC 201-204 maybe connected to a temperature controller.

For example, in the embodiment shown, TEC 201 is connected totemperature controller 221, TEC 202 is connected to temperaturecontroller 222, TEC 203 is connected to temperature controller 223, andTEC 204 is connected to temperature controller 224. In some embodiments,the temperature controllers 221-224 may be implemented using the Model3040 Temperature Controller, which is available from Newport Corporationof Irvine, Calif. In other embodiments, controllers 221-224 may consistsimply of a power amplifier.

The temperature controllers 221-224 may be interfaced with maincontroller 130. This will enable main controller 130 to control thetemperature of the different regions of device 102. Temperature controlsystem 120 may also include a temperature sensor 211 for monitoring thetemperature of inlet 109, a temperature sensor 212 for monitoring thetemperature of the PCR zone 131, a temperature sensor 213 for monitoringthe temperature of a melting zone 132, and a temperature sensor 214 formonitoring the temperature of the waste well 110. Temperature sensors211-214 may be in communication with a temperature controller and/ormain controller 130, as is illustrated in FIG. 2.

Temperature control system 120 may further include an infrared sensor250 for monitoring the temperature of the PCR zone 131 and a source ofelectromagnetic radiation 251 (e.g., a source of infrared, RF,Microwave, etc. radiation) for heating the PCR zone 131. Lastly,temperature control system 120 may include blower and heat sinks 290 forcooling one or more of TEC 201-204.

Referring now to FIG. 3, FIG. 3 illustrates imaging system 118 accordingto some embodiments of the invention. As illustrated in FIG. 3, imagingsystem 118 may include a first detector 310, a second detector 302, ablue LED 341, a red LED 342, a first laser 311, a second laser 312, anda third laser 313. Although two detectors are shown, it is contemplatedthat imaging system 118 may employ only a single detector.

Detector 310 may be configured and arranged to detect emissions (e.g.,fluorescent emissions) from PCR zone 131 and to output image datacorresponding to the detected emissions. Detector 310 may be implementedusing a conventional digital camera, such as the Canon 5D digital SLRcamera. Blue LED 341 and red LED 342 are configured and arranged suchthat when they are activated they will illuminate the PCR zone 131.

Detector 302 may be configured and arranged to detect emissions from themelting zone 132 and to output image data corresponding to the detectedemissions. Detector 302 may be implemented using a digital video camera.In one embodiment, detector 302 is implemented using an electronmultiplying charge coupled device (EMCCD).

Lasers 311-313 are configured and arranged to illuminate the meltingzone. Each laser may output a different wave length of light. Forexample, laser 311 may output light having a wave length of 488nanometers, laser 312 may output light having a wave length of 445nanometers, and laser 313 may output light having a wave length of 625nanometers.

Imaging system 118 may include a controller 330 for controlling detector310, 302 and excitation sources 341, 342, 311, 312 and 313. Controller330 may also be configured to process image data produced by thedetectors. Controller 330 may be implemented using a conventionalmicroprocessor (e.g., controller 330 may consist of a conventionalpersonal computer). Coupled to controller 330 may be an image storagedevice 331 for storing image data collected by detectors 310 and 302.Controller 330 may be in communication with main controller 130.Controller 330 may be directly connected to main controller or may beconnected to main controller through a switch 390 or other communicationdevice (e.g., an Ethernet hub).

Referring now to FIG. 4, FIG. 4 is a flow chart illustrating a process400 for amplifying and melting DNA according to some embodiments of theinvention. Process 400 may begin at 402, where a user turns on maincontroller 130, which then automatically performs system checks andinitializes other components of system 100.

In step 404, the user may create a “script” (i.e., the user may inputconfiguration information). In step 406, the user prepares device 102and places device 102 into system 100. In step 408, the user mayconfigure and/or adjust imaging system 118. In step 410, main controller130 may perform safety checks. In step 412, main controller 130 readsthe script created by the user to obtain the configuration information.

In step 414, the positioning system 194 positions well plate 196 so thatthe distal end of sipper 108 is submerged in a sample solution containedwithin one of the wells 198 of plate 196 and main controller 130 sends asignal to sample pump 114 to begin pumping. The activation of the samplepump 114 causes the sample solution to flow through channels 104. Instep 416, while the sample solution is flowing through channels 104, thetemperature of the PCR zone 131 is cycled according to the configurationinformation in the script created by the user.

In step 418, while the temperature of the PCR zone 131 is being cycled,the imaging system 118 obtains images of the PCR zone. In step 420, theimage data is processed to determine whether the DNA amplification wassuccessful. In step 422, a decision is made as to whether the PCR wassuccessful. If it was not successful, then the process may proceed tostep 424, where the user is alerted. If the PCR was successful then theprocess may proceed to step 426, where the melting zone 132 temperatureis controlled according to the script and, while the temperature isbeing controlled according to the script, images are obtained andstored. After step 426, the process may proceed back to step 416.

As FIG. 4 demonstrates, a user may use system 100 to amplify a sample ofDNA, monitor the amplification process, melt the amplified DNA, andmonitor the melting process. Thus, system 100 may provide a valuabletool for DNA analysis.

Referring now to FIG. 5, FIG. 5 is a flow chart illustrating a process500, according to some embodiments of the invention, for implementingstep 402 of process 400.

Process 500 may begin in step 501 where main controller 130 powers upother components of system 100. In step 502, main controller 130 maydetermine whether the other components are functioning appropriately. Ifit is determined that one or more other components are not functioningappropriately, the process may proceed to step 503, where maincontroller 130 informs the user that there may be a problem. Otherwise,the process may proceed to step 504.

In step 504 main controller 130 activates the upper pump 192. Thiscauses the buffer fluid 197 to fill well 198 a. In step 506 maincontroller 130 activates heat sink and blower 290. In step 508,temperature control system 120 is operated to set the reagent welltemperature to 25° C. In step 510, the temperature control system isoperated to set the waste well temperature to 25°. In step 512, thetemperature control system is operated to set the PCR zone 131 to 55° C.In step 514, the temperature control system is operated to set themelting zone 132 to 25° C. In step 516, the positioning system 194 isused to move the well plate 196 to a home position. In step 518, maincontroller 130 waits for all pressure values to be equaled to theirinitial set points.

Referring now to FIG. 6, FIG. 6 is a flow chart illustrating a process600, according to some embodiments, for implementing step 406 of process400.

Process 600 may begin in step 602, where the user fills the locusspecific reagent wells 105 and fills the common reagent well 106 andthen places the device in a device holder (not shown). In step 604 theuser may manually control the positioning system 194 to position thewell plate 196 in a desired location. For example, the user may positionwell plate 196 so that the sipper 108 is located within a desired well198 of the well plate 196.

In step 606, the user may input a desired channel pressure and mayactivate the sample pump 114. Activating the sample pump 114 causes thesample solution that is in the well 198 in which sipper 108 is locatedto enter and flow through channels 104. It also causes the reagentsolutions that are in wells 106 and 105 to enter a channel. In step 608,the user may monitor the flow of the sample solution through channels104. For example, images taken by imaging system 118 will be displayedon display device 136 and the images may show the sample solution movingthrough the channels 104.

In step 610 the user may decide whether or not to adjust the channelpressure. Adjusting the pressure of the channel will either increase ordecrease the rate at which the sample solution flows through thechannel. After adjusting the pressure, the sample pump 114 will eitherpump more or less, depending on whether the user adjusted the pressureup or down.

Referring now to FIG. 7, FIG. 7 is a flow chart illustrating a process700, according to some embodiments, for implementing step 408 of process400.

Process 700 may begin in step 702, where the user may select a filterfor detector 310, which is the detector that images the PCR zone 131. Instep 704, the user inputs into main controller 130 an identifieridentifying the selected filter. In step 706, detector 310 may obtain animage of the PCR zone 131. In step 708, the image may be displayed onthe display device 136. Along with displaying the image obtained in 706,main controller 130 may also display on the display device 136 areference image.

In step 710, the user may compare the image obtained in step 706 withthe reference image displayed in step 708. If the comparison indicatesthat the focus of detector 310 is not adjusted properly, then process700 may proceed to step 714, otherwise it may proceed to step 716. Instep 714, the user may adjust the focus of detector 310. For example,the user may move detector 310 either closer to or further away from thePCR zone 131. After step 714, process 700 may go back to step 706. Instep 716, the image obtained in step 706 may be stored and used fornormalization calculations.

In step 718, the user may position a selected filter in front ofdetector 302, which is the detector that images the melting zone 132. Instep 720, the user may turn on a selected one of the lasers 311-313 andmay adjust the output of the selected laser. In step 722, images on themelting zone 132 are obtained and displayed on the display device 136.In step 724, user may adjust the laser to intersect channels 104, ifnecessary. For example, if the images obtained in step 722 indicate thatthe laser is not properly aligned, and the user may appropriately alignthe laser so that it intersects channels 104.

In step 726, user may determine whether the focus of detector 302 isaccurate. If the focus is inadequate, process 700 may proceed to step728, otherwise it may proceed to step 730. In step 728, the user adjuststhe focus of detector 302. After step 728, the process may proceed backto step 722. In step 730, the images obtained in step 722 are stored forlater normalization calculations.

Referring now to FIG. 8, FIG. 8 is a flow chart illustrating a process800, according to some embodiments, for implementing step 414 of process400.

Process 800 may begin in step 802, where main controller 130 moves wellplate 186 to a predetermined “home” position. In step 804, maincontroller 130 sets the sample pump 114 to a predetermined pressure. Instep 806, main controller 130 reads a script created by the user todetermine which well of the well plate should be used first. In step808, main controller 130 causes the positioning system 194 to move thewell plate 196 so that a sipper 108 is placed in the well determined instep 806. In step 809 main controller 130 may determine whether the flowmode has been set to a variable flow mode or to a fixed flow mode. Ifthe flow mode has been set to be a variable flow mode then process 800may proceed to step 810, otherwise it may proceed to step 812.

In step 810, main controller 130 adjusts sample pump 114 based at leastin part on a calculation of the velocity of the sample moving throughchannels 104. This velocity calculation may be based on, among otherthings, images taken of the sample at different times as the samplemoves through the channels 104, such as, for example, as disclosed inU.S. patent application Ser. No. 11/505,358, incorporated herein byreference. In step 812, main controller 130 waits for a predeterminedamount of time. This predetermined amount of time may have been set inthe script created by the user. Immediately after the predeterminedamount of time has expired, process 800 may proceed from step 812 tostep 814. In step 814, main controller 130 causes the positioning system194 to move well plate 196 so that the sipper 108 is placed in thebuffer well 198 a.

In step 816 main controller 130 determines whether the flow mode hasbeen set to a variable flow mode or to a fixed flow mode. If the flowmode has been set to be a variable flow mode then process 800 mayproceed from step 816 to step 818, otherwise process 800 may proceedfrom step 816 to step 820. In step 818, main controller 130 adjusts thesample pump 114 based, at least in part, on a calculation of thevelocity of the sample moving through channel 104. In step 820, maincontroller 130 waits a predetermined amount of time. In step 822, maincontroller 130 determines whether another sample should be introducedinto the channels 104. If no other sample needs to be introduced intothe channels 104, then the process may proceed from step 822 to step816, otherwise the process may proceed from step 822 to step 806.

Referring now to FIG. 9, FIG. 9 is a flow chart illustrating a process900, according to some embodiments, for implementing step 416 of process400.

Process 900 may begin in step 902, where main controller 130 reads thescript created by the user to determine an initial temperature for thePCR zone 131. In step 904, the main control causes the temperature ofthe PCR zone 131 to reach the initial temperature. For example, in step904 main controller 130 may send a signal to the PCR zone temperaturecontrol system to go to the initial temperature.

In step 906, which may not be performed until after the PCR zone reachesthe initial temperature, main controller 130 causes the temperature ofthe PCR zone 131 to reach the denature stage temperature (e.g., about95° C.). In step 908, immediately after the temperature of the PCR zone131 reaches the denature temperature, main controller 130 waits for apredetermined amount of time.

In step 910, after the predetermined amount of time has elapsed, maincontroller 130 causes the temperature of the PCR zone 131 to reach theannealing stage temperature (e.g., about 55° C.). In step 912, once thetemperature of the PCR zone reaches the annealing temperature, maincontroller 130 waits for a predetermined amount of time.

In step 914, immediately after the predetermined amount of time haselapsed, main controller 130 causes the temperature of the PCR zone 131reach the extension stage temperature (e.g., about 72° C.). In step 916,main controller 130 waits for a predetermined amount of time immediatelyafter the temperature of the PCR zone reaches the extension stagetemperature. After step 916 the process may proceed to step 918, wheremain controller 130 determines whether another PCR cycle is needed ifanother PCR cycle is needed, then process 900 may proceed back to step906.

In some embodiments, step 906 includes turning on, or increasing theoutput of, source 251 while at the same time turning off, or decreasingthe heat output of, TEC 202. Also, in some embodiments, step 908includes turning off, or decreasing the output of, source 251 while atthe same time controlling TEC 202 such that the annealing temperature isreached as quickly as possible.

Referring now to FIG. 10, FIG. 10 is a flow chart illustrating a process1000, according to some embodiments, for implementing step 418 ofprocess 400.

Process 1000 may begin in step 1002, where a determination is made as towhether the image trigger point has been reached. In some embodimentsthe image trigger point is reached at the point in time when the PCRcycle begins the extension stage.

In step 1004, once the trigger point has been reached, main controller130 may wait for a predetermined amount of time to allow the temperatureto stabilize, which predetermined amount of time may have been specifiedin the script created by the user.

In step 1006, main controller 130 selects one of the LEDs 341 or 342based on the script and turns on the selected LED for a predeterminedamount of time, which amount of time may be specified in the script, andthen immediately turns off the selected LED after the predeterminedamount of time has expired.

In step 1008, controller 340 may configure the PCR zone 131 detector(e.g., detector 310) according to the script. For example, controllermay set the detectors aperture and shutter speed.

In step 1010, detector 310 is used to acquire an image of the PCR zone131. In step 1012 the image is saved. In step 1014, main controller 130determines whether two colors are required. If two colors are required,then the process may proceed from step 1014 to step 1016, otherwise theprocess may proceed from step 1014 back to step 1002.

In step 1016, main controller 130 selects a different LED than the oneselected in step 1006 and then turns on the selected LED for apredetermined amount of time and then immediately turns off the LEDafter the predetermined amount of time has expired. After step 1016,process 1000 may proceed to step 1008.

Referring now to FIG. 11, FIG. 11 is a flow chart illustrating a process1100, according to some embodiments, for implementing step 426 ofprocess 400.

Process 1100 may begin in step 1102, where the temperature profile forthe melting process is determined. For example, the temperature profilemay include a maximum temperature, a minimum temperature and a ramprate. In step 1104, melting zone 132 is heated according to thedetermined temperature profile. For example, main controller 130 maycause the temperature control system 120 to cause the temperature of themelting zone 132 to reach the minimum temperature and, once the minimumtemperature is reached, main controller 130 may cause the temperaturecontrol system 120 to increase the temperature of the melting zone tothe maximum temperature at a rate equal to the ramp rate.

In step 1106, an image of the melting zone 132 is captured and displayedon the display device 136. FIG. 12 illustrates an image 1201 that may bedisplayed using display device 136 in step 1106. Each dot 1202represents a channel 104 (i.e., dot 1202 a represents channel 104 a anddot 1202 b represents channel 104 b).

In step 1108, after the image is displayed, the user may specify aregion of interest for each channel 104. This step is illustrated inFIG. 13, which shows a region of interest 1302 for each channel.

In step 1110, after the user specifies the regions of interest, maincontroller 130 may determine a pixel window 1402 (see FIG. 14) thatcontains all of the regions of interest. In some embodiments, the pixelwindow is the smallest rectangular pixel region of the sensor ofdetector 302 that includes all of the regions of interest.

In step 1112, the detector 302, which images melting zone 132, isoperated so that it can take several images per second (e.g., at leastabout 10 images per second), but, for each image, record only the pixelsthat are included in the pixel window.

In step 1114, the regions of interest that were defined by the user areextracted from the recorded image data. In step 1116, the fluorescenceintensity for each region of interest is calculated. In step 1118, maincontroller 130 plots relative fluorescence units vs. temperature. Instep 1120, main controller 130 plots the derivative vs. temperature. Instep 1122, a determination is made as to whether the melting process iscomplete. If the melting process is not complete the process may proceedback to step 1112, otherwise the process may proceed to step 416 ofprocess 400.

While various embodiments/variations of the present invention have beendescribed above, it should be understood that they have been presentedby way of example only, and not limitation. Thus, the breadth and scopeof the present invention should not be limited by any of theabove-described exemplary embodiments.

Additionally, while the processes described above and illustrated in thedrawings are shown as a sequence of steps, this was done solely for thesake of illustration. Accordingly, it is contemplated that some stepsmay be added, some steps may be omitted, and the order of the steps maybe re-arranged.

1. A system for performing real-time PCR and melting of DNA, comprising: a microfluidic device comprising: a microfluidic channel having a PCR zone and a DNA melting zone, a first reagent well in fluid communication with the channel and a second reagent well in fluid communication with the channel; a sipper connected to the microfluidic device, the sipper being in fluid communication with the channel; a pump for forcing a sample to flow through the channel; a well plate comprising a buffer well for storing a buffer solution and a sample well for storing a sample solution containing a DNA sample; a buffer solution storage container for storing a buffer solution, the buffer solution storage container being in fluid communication with the buffer well of the well plate; a positioning system operable to position the well plate; a temperature control system for cycling the temperature of a sample while the sample is in the PCR zone of the channel and for providing heat for melting the DNA contained in the sample while the sample is in the DNA melting zone; an imaging system for detecting emissions from the PCR zone and from the DNA melting zone, the imaging system comprising: a first excitation source and a detector configured and arranged to detect emissions from the PCR zone and a second excitation source and a detector configured and arranged to detect emissions from the DNA melting zone; wherein the first excitation source for the PCR zone and the second excitation source for the DNA melting zone are different types; and a main controller in communication with (a) the temperature control system, (b) the positioning system, (c) the imaging system, and (d) a display device.
 2. The system of claim 1, wherein the positioning system comprises a positioner.
 3. The system of claim 2, wherein the positioning system further comprises a stepping drive operable to drive the positioner.
 4. The system of claim 3, wherein the positioning system further comprises a controller for controlling the stepping drive.
 5. The system of claim 1, wherein the imaging system comprises: a first light emitting diode directed to the PCR zone of the channel, a second light emitting diode directed to the PCR zone of the channel, and a laser directed to the melting zone of the channel.
 6. The system of claim 5, wherein the first light emitting diode and the second light emitting diode are different colors, and wherein the first and second light emitting diodes are operated independently by the main controller.
 7. The system of claim 1, wherein the second detector comprises a electron multiplying charge coupled device (EMCCD).
 8. The system of claim 1, wherein the imaging system further comprises a detector controller interfaced with the first detector and the second detector.
 9. The system of claim 8, wherein the detector controller is communicatively connected to the main controller.
 10. The system of claim 9, wherein the detector controller is connected to the main controller through an Ethernet hub.
 11. The system of claim 1, wherein the temperature control system comprises a first heater configured to heat a sample within the PCR zone and a second heater configured to heat a sample within the DNA melting zone.
 12. The system of claim 11, wherein the first heater comprises a first thermoelectric cooler (TEC) and the second heater comprises a second thermoelectric cooler (TEC).
 13. The system of claim 12, wherein the temperature control system further comprises a first temperature controller coupled to the first TEC and a second temperature controller coupled to the second TEC.
 14. The system of claim 13, wherein the first temperature controller is coupled to the main controller and the second temperature controller is coupled to the main controller.
 15. The system of claim 14, wherein the first temperature controller consists of a power amplifier.
 16. The system of claim 11, wherein the temperature control system further comprises a source of electromagnetic radiation, which source is arranged to illuminate the PCR zone.
 17. The system of claim 11, wherein the first or second heater is configured to produce infrared radiation.
 18. The system of claim 1, wherein the main controller monitors the sample during PCR amplification.
 19. The system of claim 1, wherein the main controller monitors the sample during DNA melting.
 20. The system of claim 1, wherein the main controller adjusts one or more of the imaging system, the temperature control system, the positioning system or the pump.
 21. The system of claim 1, wherein the main controller triggers one or more additional PCR cycles in response to the data received from the imaging system.
 22. The system of claim 1, wherein the main controller triggers one or more additional DNA melts in response to the data received from the imaging system.
 23. The system of claim 1, wherein the main controller is configured to operate the first excitation source and the second excitation source independently, and wherein the main controller is configured to operate the second excitation source in response to analysis of the emissions detected by the PCR zone detector.
 24. A system for performing real-time PCR and melting of DNA, comprising: a microfluidic device comprising: a microfluidic channel having a PCR zone and a DNA melting zone, a first reagent well in fluid communication with the channel and a second reagent well in fluid communication with the channel; a sipper connected to the microfluidic device, the sipper being in fluid communication with the channel; a pump for forcing a sample to flow through the channel; a well plate comprising a buffer well for storing a buffer solution and a sample well for storing a sample solution containing a DNA sample; a buffer solution storage container for storing a buffer solution, the buffer solution storage container being in fluid communication with the buffer well of the well plate; a positioning system operable to position the well plate; a temperature control system for cycling the temperature of a sample while the sample is in the PCR zone of the channel and for providing heat for melting the DNA contained in the sample while the sample is in the DNA melting zone; an imaging system for detecting emissions from the PCR zone and from the DNA melting zone, the imaging system comprising: a first excitation source comprising a laser emitting diode and a detector configured and arranged to detect emissions from the PCR zone and a second excitation source comprising a laser and a detector configured and arranged to detect emissions from the DNA melting zone; a main controller in communication with (a) the temperature control system, (b) the positioner, (c) the imaging system, and (d) a display device. 